The present invention relates to rapid methods for detecting microorganisms in products for human consumption or use, and more particularly, but not by way of limitation, to rapid methods for detecting the presence or absence of total coliform bacteria, E. coli or thermotolerant coliform bacteria in milk products.
Protection from deleterious microbial contaminants is a global issue. Each year millions of people throughout the world become ill, and thousands die, from contaminated food and water. Disturbing newspaper headlines and stories of epidemic and endemic diseases have increased public awareness of these problems. Testing for bacteria has thus received increasing attention from consumers and public regulatory bodies. In view of this, there is a growing demand for faster methods of detecting microbial contamination. The constant media attention on severe health risks related to microbial contamination of products consumed by humans is leading to increased consumer awareness and public regulatory pressure regarding the safety and the quality of food, water and pharmaceutical products. In addition, economic forces are urging companies to reduce costs by reducing waste, processing time and stock levels.
Several incidents of food borne bacteria outbreaks have increased consumer awareness regarding the safety of and the exact contents of food products. Recent examples of microbial contamination receiving major public attention include:
The Japanese E. coli outbreak, May 1996. The severe outbreak of infections from the bacterium E. coli 0157:H7 in Osaka, Japan, caused the death of 8 and seriously sickened over 9,000. The source is believed to be radish sprouts from one single supplier. PA1 The Scottish E. coli epidemic, 1996. The E. coli outbreak in Lanarshire, Scotland in 1996, created vast political turmoil and investigation after the deaths of 18 and hundreds affected from contaminated meat. PA1 Norway, summer 1997: Alarmingly high numbers of E. coli bacterium (including a less aggressive type of the 0157 bacterium) was found in 800 tonnes meat from a Norwegian abattoir. This incident could have incurred large casualties. PA1 Hudson Hamburger Contaminants, USA 1997. Twenty-five million pounds of hamburger meat were recalled and destroyed following the detection of pathogenic E. coli in samples of the meat. PA1 (a) combining the original sample with an actuating medium having a fat emulsifying composition and a fluorogenic substrate which when metabolized yields a fluorescent product, preferably 4-methylumbelliferone; PA1 (b) incubating the combined sample and actuating medium mixture at a temperature preferred for incubating total, thermotolerant, or E. coli coliform cells, for a predetermined duration; PA1 (c) adjusting the pH of the incubated combined sample to an alkaline pH and irradiating said sample with a predetermined excitation wavelength of light; PA1 (d) measuring a fluorescence value from the irradiated combined sample; and PA1 (e) concluding that the original sample is contaminated with the specified bacteria when the fluorescence value equals or exceeds a predetermined threshold value which corresponds to a particular concentration value of the fluorescent product.
It is estimated that the industrial market for detection of microbial contaminants was approximately 600 million tests in 1997, amounting to a value of approximately USD 2.5 billion. Of the tests performed annually, the food segment is by far the largest segment, with approximately 310 million tests (53%), followed by the pharmaceutical segment with approximately 200 million tests (32%), the beverage segment with approximately 60 million tests (10%) and finally the environmental segment with approximately 30 million tests (5%). More than 80% of today's testing is performed with slow traditional methods (giving results in 2-3 days), which are laborious and expensive to use. These methods typically use agar plates or standard pour plates (plastic dishes with a nutrient medium), enhancing bacterial growth so that they multiply and their presence can be identified visually as colonies and counted. It is expected that the need for more effective measurements will lead to a significant conversion from slower traditional methods to more rapid and easy to use methods over the next 5 to 10 years. The total market is expected to exceed 800 million tests by 2005, and it is believed that rapid methods will represent 30-40% of the market.
Traditional microbiological methods, which take 18-72 hours to generate results, have led existing regulations to focus on testing of finished products. However, sampling from end product batches for testing does not guarantee that all products in one batch are of good quality. Food processing involves a number of steps and hand-overs (e.g. from the abattoir to the fast food restaurant), giving multiple operations and points for potential microbial carry-over and contamination. The nature of end product testing can therefore not capture every incident of microbial contamination. The ability to rapidly test for contamination at various steps early in a production line would minimize the chances that entire batches of products would have to be destroyed, as is often the case when only end point testing is carried out.
However, with the demand for "just-in-time" deliveries, few companies are able to wait for results of microbial testing. Traditional test methods therefore have value only for historical and documentation purposes. Some producers, however, hold goods until test results are complete, thus raising stock costs. The ability to provide "real time" information for the factories, avoiding contaminated products being shipped, reducing wastage and stocks is therefore desired.
Manufacturers who fail to deliver safe and high quality food products face severe problems, like reduced brand name value, loss or sales, product liability suits and, in worst case, plant closures. The retail industry has increasingly adopted private labels in shelves. The risk of bad publicity and loss of sales in case of "food poisoning" from their branded products, leads retailers to request documentation or testing and implementation of microbial quality control systems from their suppliers. This puts pressure for increased quality control throughout the entire product chain, from delivery of raw materials, through processing, to the end-products.
Over the last 20 years, some new and "easy to use" methods (such as COLILERT and 3M PETRIFILM) have been introduced and have gained approximately 15% of the total market today. These methods are different from the traditional methods in that they have made daily laboratory work easier by reducing many of the practical steps operators take when conducting microbial tests. However, the detection time for these methods, although down from 2-3 days, is still about one day. This is still too long for products that are finished and already shipped to customers. These new tests have therefore not significantly altered how and where companies perform their routine tests.
COLILERT is a 24 hour growth-based method for detection of coliforms/E. coli in drinking water. The product has gained widespread usage in the U.S. PETRIFILM, by 3M, represents another product targeted at making microbiology measurements easier to do for workers. Petrifilm is similar to traditional methods regarding time to results and reading of results but eliminates or minimizes sample and media preparations. This is an advance and makes results much more consistent.
Also, in the last 3-4 years, a new class of rapid tests for microbial contamination has managed to gain a market share of approximately 5%, amounting to 30 million tests. Food processing plants must routinely stop production to clean and sanitize the facility. In many plants this occurs during the night, before the plant begins production in the morning. Plant quality control analysts have been perplexed about how to determine if the plant is properly sanitized.
An effective HACCP (Hazard Analysis Critical Control Point) program is dependent upon access to rapid and easy-to-use sanitation screening tests, especially in early states in the production process.
Healthy animals carry pathogens for humans in their intestines and on their hide and hooves. Slaughter unavoidably disseminates these pathogens to the carcass. Excision is considered the most effective bacterial sampling method, but in red meat processing facilities excision is neither practical nor acceptable. Consequently a more practical, non-destructive, and rapid method for carcass bacterial sampling must be validated. These factors should be accomplished without significantly affecting the total sum of recovered bacteria.
Traditional methods for assaying bacteria on surfaces are based on swabbing the surface followed by either a culture of the swab in a media that supports growth or by rinsing swab in a buffer and plate on agar and incubation for 24-48 hours. Other methods are to use contact plates or petrifilm to press on the surface, and then incubate for 24 to 72 hours. (The disadvantage with the contact plates and petrifilm is that they cannot be used on wet surfaces, and they may leave some of the growth medium on the carcass, enhancing bacterial growth).
To satisfy this need rapid (5 minutes) tests measuring ATP (adenosine triphosphate), a biological molecule which is present in among all living microorganisms, have been developed. The test can determine whether a facility has been properly cleaned and sanitized. It is also a very easy to use test, so it can be incorporated into the job of the cleaning crew and does not have to be performed by laboratory technicians. However, the ATP test does not specifically detect bacterial contamination, only the presence of organic materials which contain ATP.
The purpose of microbial testing is mainly to identify the presence and risk of presence of bacteria dangerous to the human body. In many cases the level of contamination must also be measured, and in certain cases the microbes must be identified. Microbial tests typically cover either one specific bacterium, or a limited spectrum of bacteria. They are also often limited to testing of specific substances (e.g. water, meat, surfaces).
Specific pathogens are difficult and time-consuming to detect, often taking several days. Hence, indicators of the presence of pathogens, such as coliforms are preferred for analysis and monitoring of water and food quality.
Indicator testing for Total Viable Organisms (referred to TVO or TVC) and coliforms are the most widely used tests for routine monitoring of microbial contamination. Microbial testing and technology requirements vary widely across industries.
The environmental industry segment is concerned with the monitoring of water quality in drinking water, and bathing water (e.g., spas and swimming pools) manufacturing process water, and ambient/recreational water. The global market consists of approximately 30 million tests, mainly for coliforms/E. coli in drinking water. Routine testing of drinking water has traditionally been enforced by stringent public regulatory requirements in every country.
The non-alcoholic beverage industry consists of the bottled water, stilled soft drinks, carbonated soft drinks, and beer production segments. The majority of coliform tests in the beverage industry are performed on bottled water. Larger bottled water producers acknowledge that more rapid results would help them reduce stock levels and potential costs related to calling back shipped products.
The food processing industry consists of a number of products, including milk and dairy products, meat, fish agriculture and multiple food manufacturing products, with different regulatory and company requirements for microbial testing. The industry currently demands a range of technologies to accommodate its testing needs, comply with new regulations, enhance food safety and reduce costs associated with laboratory testing, processing times and stock levels.
Standard testing procedures in the milk industry currently include the following:
(1) International Dairy Federation (IDF) 73A:1985, Milk and Milk Products, Enumeration of coliforms--colony count technique and most probable number at 30.degree. C.;
(2) International Standard, ISO, 5541/1 Milk and milk products, Enumeration of coliforms--Part 1: Colony count technique and most probable number at 30.degree. C. First ed. Dec. 1, 1986;
(3) International Standard, ISO, 5541/2 Milk and milk products, Enumeration of coliforms--Part 2: Most probable number at 30.degree. C. First ed. Dec. 1, 1986; and
(4) International Standard, ISO, 11866/3 Milk and milk products, Enumeration presumptive Escherichia coli--Part 3: Colony count technique at 44.degree. C. using membranes. First ed. Feb. 15, 1997.
Unfortunately, these methods generally take at least 48 to 72 hours to obtain results.
The pharmaceutical industry performs approximately 200 million tests annually and requires the highest standard of microbial quality. Pharmaceutical producers are seeking better control of incoming raw materials, processing stages and final products.
The food service industry (such as caterers and fast food restaurants) is pushing suppliers to document quality of delivered products. Today routine bacteria tests are mostly performed at external laboratories. However, there is reason to believe that recent severe incidents of microbial contamination, leading to food-borne disease outbreaks and fatalities have lead the food service industry to reevaluate its quality assurance systems. It is believed that giving caterers the possibility of near-real-time test for specific bacteria indicators like coliforms (as opposed general tests which detect ATP), in the form of a simple instrument test would help companies secure the quality of their sanitation process and incoming products.
As evident from the above, there continues to be a need for methods which will rapidly detect the presence of total coliform bacteria, thermotolerant (including fecal) coliform bacteria or E. coli or pathogenic E. coli 0-157 in samples.